Compositions and methods for altering human cutaneous microbiome to increase growth of staphylococcus epidermidis and reduce staphylococcus aureus proliferation

ABSTRACT

An antibacterial composition comprising arginine bicarbonate, zinc carbonate, preferably arginine bicarbonate and zinc carbonate (ABZC), in combination, plus one or more physiologically acceptable excipients, administered for the modification of cutaneous microfloras, generally to inhibit the growth of pathogenic  Staphylococcus aureus  bacteria by promoting the growth of non-pathogenic  Staphylococcus epidermidis  bacteria.

FIELD OF THE INVENTION

The present invention relates to compositions and methods forselectively increasing the growth of Staphylococcus epidermidis andinhibiting the growth of Staphylococcus aureus bacteria in the cutaneousmicrobiome. More particularly, the present invention relates tocompositions and methods for increasing the growth of Staphylococcusepidermidis and reducing the incidence of MRSA and MSSA by the selectiveinhibition of Staphylococcus aureus.

BACKGROUND OF THE INVENTION

The cutaneous microbiome in humans is comprised of a variety ofmicroorganisms, of which staphylococci, corynebacteria andpropionibacteria are among the most prominent (Starkemann et al., 2005,Troccaz et al., 2004, Jackman, 1982). These bacteria act upon odorlessprecursors contained in sweat per se, producing sugars, sugar amines,amino acids, and short chain carboxylic acids (SCCAs), of which some aredegraded further to products that include odorants that are associatedto a major extent with cutaneous odor (Zeng et al, 1991; Jackman, 1982).

One frequent undesirable member of the cutaneous microbiome,Staphylococcus aureus (Staph. aureus, including methicillin-resistantStaph. aureus (MRSA) and methicillin-susceptible Staph. aureus (MSSA)),has a well-known role in invasive infections in humans. It is one of themost problematic of human pathogens, because it is capable of wideinfection and fatalities (see, e.g., David et al., 2010, Mainous III etal., 2006, Klevens et al., 2007). Antibiotics used against it haveachieved limited success. Methicillin is effective but limited becauseof adaptation, which can result in the emergence of MRSA, which isrepresentative of antibiotic failure occurring now more so withincreasing frequency of use (see, e.g., David et al 2010, Chen et al2006, Centers for Disease Control and Prevention 2003).

SUMMARY OF THE INVENTION

The present invention is directed to compositions of zinc salts andarginine and/or its salts for the selective inhibition of Staph. aureusgrowth and favoring growth of Staph. epidermidis.

The present invention is directed to a topical antibacterial compositionincluding arginine or its salt, a zinc salt, and, optionally, a bufferfor maintaining the pH of the composition at 6.0 or greater. Theantibacterial compositions of the invention are useful in selectivelyinhibiting the growth of Staphylococcus aureus and increasing the growthof Staphylococcus epidermidis bacteria in the cutaneous microbiome.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying figures, inwhich:

FIG. 1 is a graph showing the effect of arginine bicarbonate on growthof 8.3% (v/v) Staphylococcus epidermidis incubated with 12.0 mM zinccarbonate, 6.0 mM CIL and 24.0 mM arginine bicarbonate at 37° C. for 72hours.

FIG. 2 is a graph showing the effect of arginine bicarbonate on growthof 8.3% (v/v) Staphylococcus aureus (MSSA), compared to 8.3% (v/v)Staphylococcus epidermidis, incubated with 12.0 mM zinc carbonate, 6.0mM CIL and 24.0 mM arginine bicarbonate at 37° C. for 72 hours.

FIG. 3 is a graph showing the effect of arginine bicarbonate on growthof an 8.3% (v/v) 1:1 mixture of Staphylococcus epidermidis andStaphylococcus aureus (MSSA) incubated with 12.0 mM zinc carbonate and6.0 mM CIL, with or without 24.0 mM arginine bicarbonate, at 37° C. for72 hours.

FIG. 4 is a graph showing the effect of arginine bicarbonate on growthof 8.3% (v/v) Staphylococcus aureus (MSSA) incubated with 12.0 mM zinccarbonate and 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate,at 37° C. for 72 hours.

FIG. 5 is a graph showing the effect of arginine bicarbonate on growthof 8.3% (v/v) Staphylococcus epidermidis incubated with 12.0 mM zinccarbonate and 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate,at 37° C. for 72 hours.

FIG. 6 is a graph showing the effect of zinc carbonate on growth of 8.3%(v/v) Staphylococcus aureus (MSSA) incubated with 12.0 mM zinc carbonateand 6.0 mM CIL at 37° C. for 72 hours.

FIG. 7 is a graph showing the effect of arginine bicarbonate on the pHand growth of 8.3% (v/v) Staphylococcus aureus (MSSA), 8.3% (v/v)Staphylococcus epidermidis or an 8.3% (v/v) 1:1 mixture ofStaphylococcus aureus (MSSA) and Staphylococcus epidermidis incubatedwith 12.0 mM zinc carbonate and 6.0 mM CIL, with or without 24.0 mMarginine bicarbonate, at 37° C. for 72 hours.

FIG. 8 is a graph showing the effect of arginine bicarbonate on thegrowth of 8.3% (v/v) Staphylococcus epidermidis or 8.3% (v/v)Staphylococcus aureus (MRSA) incubated with 12.0 mM zinc carbonate, 6.0mM CIL and 24.0 mM arginine bicarbonate at 37° C. for 72 hours.

FIG. 9 is a graph showing the effect of arginine bicarbonate on thegrowth of 8.3% (v/v) Staphylococcus aureus (MRSA), compared to 8.3%(v/v) Staphylococcus epidermidis, incubated with 12.0 mM zinc carbonateand 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate, at 37° C.for 72 hours.

FIG. 10 is a graph showing the effect of arginine bicarbonate on thegrowth of 8.3% (v/v) Staphylococcus epidermidis or 8.3% (v/v)Staphylococcus aureus (MRSA) incubated with 12.0 mM zinc carbonate, 6.0mM CIL and 24.0 mM arginine bicarbonate, and modified versions of thismedium, at 37° C. for 72 hours.

FIG. 11 is a graph showing the effect of arginine bicarbonate on thegrowth of an 8.3% (v/v) 1:1 mixture of Staphylococcus aureus (MRSA) andStaphylococcus epidermidis incubated with 12.0 mM zinc carbonate and 6.0mM CIL, with or without 24.0 mM arginine bicarbonate, at 37° C. for 72hours.

FIG. 12 is a graph showing the effect of arginine bicarbonate on thegrowth of 8.3% (v/v) Staphylococcus aureus (MRSA) incubated at variousdilutions with 12.0 mM zinc carbonate and 6.0 mM CIL, with or without24.0 mM arginine bicarbonate, at 37° C. for 72 hours.

FIG. 13 is a graph showing the effect of arginine bicarbonate on thegrowth of 8.3% (v/v) Staphylococcus epidermidis incubated at variousdilutions with 12.0 mM zinc carbonate and 6.0 mM CIL, with or without24.0 mM arginine bicarbonate, at 37° C. for 72 hours.

FIG. 14 is a graph showing the effect of zinc carbonate on the growth of8.3% (v/v) Staphylococcus aureus (MRSA) incubated at various dilutionswith 12.0 mM zinc carbonate and 6.0 mM CIL at 37° C. for 72 hours.

FIG. 15 is a graph showing the pH responses of 8.3% (v/v) Staphylococcusepidermidis, 8.3% Staphylococcus aureus (MRSA) or an 8.3% (v/v) 1:1mixture of Staphylococcus epidermidis and Staphylococcus aureus (MRSA)to 12.0 mM zinc carbonate and 6.0 mM CIL, with or without 24.0 mMarginine bicarbonate, at 37° C. for 72 hours.

FIG. 16 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of 8.3% (v/v) Staphylococcus epidermidis or 8.3%(v/v) Staphylococcus aureus (MSSA) incubated with 12.0 mM zinccarbonate, 6.0 mM CIL and 24.0 mM arginine bicarbonate, at 37° C. for 72hours.

FIG. 17 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of an 8.3% (v/v) 1:1 mixture of Staphylococcusepidermidis and Staphylococcus aureus (MSSA) incubated with 12.0 mM zinccarbonate and 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate,at 37° C. for 72 hours.

FIG. 18 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of an 8.3% (v/v) 1:1 mixture of Staphylococcusepidermidis and Staphylococcus aureus (MSSA) incubated with 12.0 mM zinccarbonate and 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate,at 37° C. for 72 hours.

FIG. 19 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of 8.3% (v/v) Staphylococcus epidermidis or 8.3%(v/v) Staphylococcus aureus (MRSA) incubated with 12.0 mM zinccarbonate, 6.0 mM CIL and 24.0 mM arginine bicarbonate, at 37° C. for 72hours.

FIG. 20 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of 8.3% (v/v) Staphylococcus epidermidis or 8.3%(v/v) Staphylococcus aureus (MRSA) incubated with 12.0 mM zinc carbonateand 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate, at 37° C.for 0 hour.

FIG. 21 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of 8.3% (v/v) Staphylococcus epidermidis or 8.3%(v/v) Staphylococcus aureus (MRSA) incubated with 12.0 mM zinccarbonate, 6.0 mM CIL and 24.0 mM arginine bicarbonate, and modifiedversions of this medium, at 37° C. for 24 hours.

FIG. 22 is a photograph showing the effect of 24.0 mM argininebicarbonate on growth of an 8.3% (v/v) 1:1 mixture of Staphylococcusepidermidis and Staphylococcus aureus (MRSA) incubated with 12.0 mM zinccarbonate and 6.0 mM CIL, with or without 24.0 mM arginine bicarbonate,at 37° C. for 48 hours.

FIG. 23 is a photograph showing the effect of arginine bicarbonate ongrowth of an 8.3% (v/v) 1:1 mixture of Staphylococcus epidermidis andStaphylococcus aureus (MRSA) incubated with 12.0 mM zinc carbonate, 6.0mM CIL and 24.0 mM arginine bicarbonate, with or without an additional24 mM arginine bicarbonate added at indicated times, at 37° C. for 72hours,

DETAILED DESCRIPTION OF THE INVENTION

Corynebacteria, staphylococci and proprionibacteria are among the mainmicroorganisms present in the cutaneous microbiome, with Staph.epidermidis, C. striatum and P. avidum as prominent representativebacteria

Unexpectedly, it has been discovered that certain compositions includinga zinc salt and arginine and/or its salt are useful as antibacterialcompositions, inhibiting Staph. aureus growth while favoring Staph.epidermidis growth. This ability to select between Staph. aureus andStaph. epidermidis allows the treatment of significant physiological andhealth-related disease conditions caused by aberrant or excessive growthof Staph. aureus (see, e.g., Peacock et al., 2001, Uehara et al., 2000).Although Staph. aureus is capable of wide infection and fatalities (see,e.g., David et al., 2010, Mainous III et al., 2006, Klevens et al.,2007), current antibiotic treatments have achieved limited success dueto the emergence of resistant Staph. aureus strains, e.g., MRSA (see,e.g., David et al 2010, Chen et al 2006, Centers for Disease Control andPrevention 2003). A recent discovery has shown that firmicidin (Gallo etal., 2013, Nakatsuji et al., 2012), a newly discovered antibioticgenerated by Staph. epidermidis, can reduce Staph. aureus, but it is notknown whether this will, like other antibiotics, succumb to adaptationand loss of effectiveness. From a commercial stand-point, this approachis likely to be costly.

Unlike traditional antibacterial treatments, the compositions of thepresent invention are aimed at modulating natural interactions betweenStaph. aureus and other prominent members of the cutaneous microflora,e.g., Staph. epidermidis (see, e.g., Frank et al., 2010, Vehara et al.,2000, Wertheim et al., 2005). These bacteria naturally compete, e.g.,for local resources and attachment to mucosal sites (Frank et al.,2010). The compositions of the invention, rather than merely targetingStaph. aureus, render an ecological change that favors selection ofdesirable Staph. epidermidis over non-desirable Staph. aureus bacteria.

Because the compositions of the present invention derive theirantibacterial effectiveness not only by targeting Staph. aureusdirectly, but also by enhancing the ability of other, non-pathogenicbacteria (e.g., Staph. epidermidis) to out-compete Staph. aureus. Thecompositions disclosed here are less likely to be susceptible to theemergence of resistant strains (e.g., MRSA) than traditionalantibacterial treatments.

A further advantage of the present invention is that the compositionsdisclosed herein are effective in reducing cutaneous odor production.Thus, a single topical composition may be used as both deodorant andantibacterial treatment.

Antibacterial compositions as described herein are administered,preferably topically, for the treatment of any one or more symptomsdesirable of change, e.g., Staph. aureus growth. Dosage forms are solidor free-flowing. Dosage forms include, but are not limited to, soaps,sprays, drops, aerosols, powders, roll-ons, lotions, creams, sticks,solutions, sachets, colloidal suspensions, films, patches and ointments.

Antibacterial compositions as described herein may have a pH of at least6.0, or at least 7.0, or at least 8.0, or at least 9.0 upon topicaladministration.

Antibacterial compositions as described herein may optionally includeone or more physiologically acceptable buffers sufficient to maintainthe pH of said composition, e.g., at 6.0 or greater, at 7.0 or greater,at 8.0 or greater, or at 9.0 or greater upon topical application. Suchbuffers are generally known in the art, and may include, e.g., ACES,acetic acid, ADA, AMP, AMPD, bicine, bis-Tris, bis-Tris propane, BES,boric acid, cacodylate, CABS, CAPS, CAPSO, CHES, citric acid,diethanolamine, DIPSO, EPPS/HEPPS, ethanolamine, formic acid, glycine,glycylglycine, HEPES, HEPPSO, histidine, imidazole, lactic acid, maleicacid, malic acid, MES, MOPS, MOPSO, morpholine, phosphate, phosphoricacid, picolinic acid, PIPES, piperazine, piperidine, pivalic acid,POPSO, pyridine, succinic acid, TAPS, TAPSO, TEA, TES, tricine, and/orTris.

Except where otherwise noted, the terms “axillary odor” and “foot odor”are used interchangeably herein, the terms “microbiome,” “microbiota,”and “microflora” are used interchangeably herein, the terms “foot,”“foot web,” “foot-web,” “toe,” “toe web” and “toe-web”are usedinterchangeably herein, and the terms “odor” and “malodor” are usedinterchangeably herein.

The terms “cutaneous” and “skin” refer, in the context of the presentinvention, regions of the human body including, e.g., the axilla,foot-webs and nasal atrium.

The terms “physiologically acceptable” and “physiologically-acceptable”denote, in the context of the present invention, “safe and effectivewhen administered to humans and/or mammals in need thereof,” e.g., toreduce axillary odor, promote the growth of Staphylococcus epidermidisbacteria, inhibit the growth of Staphylococcus aureus bacteria, or anyor all of the preceding.

Examples

The following examples are intended to illustrate, but not limit, thepresent disclosure.

Growth of Staph. aureus (MSSA or MRSA) and Staph. epidermidis when oneor the other or a mixture of the two bacteria were incubated in thepresence of (i) cysteine and (ii) isoleucine, leucine, phenylalanine.Zinc carbonate was also provided with and without arginine bicarbonateat 37° C. for 72 hours and with additional above ingredients adding intothe cultural media in 37° C. water bath in 24 and 48 hours.

Materials and Methods for Growth Comparison Experiments Between Staph.epidermidis and Staph. aureus

-   -   (a) Preparation of Agar plates containing various bacterial        growth media. Preparation included (i) BHI Blood agar (Fisher        Scientific, Springfield, N.J. USA) and (ii) CHROMagar Staph.        aureus agar (CHROMagar, Paris, France), especially prepared for        the isolation and identification of Staph. aureus; if present,        it results in colonies that show a characteristic mauve color        that enables ease of identification (French, 2009, Han et al.,        2007).    -   (b) Stock solutions of CIL amino acids. These amino acids        include cysteine, isoleucine and leucine with each present at a        concentration of 72 mM. Aqueous solutions of each were        sterilized by syringe filtering as described earlier (Zhang and        Kleinberg, 2014).    -   (c) Stock aqueous solutions of arginine bicarbonate at 144 mM        and zinc carbonate at 72 mM. Stock solutions of 144 mM arginine        bicarbonate were sterilized together with 72 mM zinc carbonate        by syringe filtering. Zinc carbonate has a limited solubility        and hence is sterilized by first autoclaving as a powder and        then dissolving it until saturation in sterile distilled water        is achieved. This means that at 72 mM and above, it may have to        be used as a zinc carbonate suspension.    -   (d) Rabbit coagulase plasma (PL 850) and Prolex Staph Xtra Latex        kits (PL.1080). Both of these items are provided as a kit and        are obtained from Pro-Lab Diagnostics, Austin, Tex. They are        prepared for the identification of pathogenic staphylococci        (e.g., Staph. aureus).    -   (e) Experimental and control incubation mixtures containing        Staph. epidermidis (ATCC 12228) and Staph. aureus (MSSA and/or        MRSA). These incubation mixtures were prepared for comparison        purposes and included MSSA (ATCC 25923) or MRSA (ATCC 33591)        bacterial species mixed with the microorganism Staph.        epidermidis. Pure cultures of Staph. epidermidis and Staph.        aureus (MSSA or MRSA) were each prepared as 25% (v/v) bacterial        suspensions in sterile distilled water. As above and as much as        possible, bacterial pellets were broken up into fine particles,        by stirring with a sterile TB syringe and a 25-27 gauge needle,        if and when needed.

As a preparatory step, the resulting suspensions obtained were incubatedin a shaking water bath at 37° C. for one hour, in order to depletestored substrates acquired by some bacteria, during their preparatorygrowth period (Wijeyeweera and Kleinberg, 1989). The pH of each of theabove bacterial suspensions was then measured by transferring 0.25 ml ofsuch to a small sterile test-tube and measuring its pH. This made iteasier to avoid any bacterial contamination during handling. Sampleswere then stored at 4° C. until time of inoculation of agar plates.

Preparation of Experimental and Control Samples

Preparation was performed according to information in Table 1 below.

TABLE 1.1 Experimental (A and B) and negative control (C) samples wereprepared according to the following ABC Composition Tables: A.Experimental samples (ml) Composition I II III IV V VI Finalconcentrations Amino acids Cys 72 mM 0.225 0.225 0.225 0.225 0.225 0.2256 mM Ieu 72 mM 0.225 0.225 0.225 0.225 0.225 0.225 6 mM Ileu 72 mM 0.2250.225 0.225 0.225 0.225 0.225 6 mM Zinc Carbonate (72 mM) 0.45 0.45 0.450.45 0.45 0.45  12 mM  Arg. Bicarbonate (144 mM) 0.45 0.45 0.45 — — — 24mM (IV, V, VI = 0 mM) Staph. epidermidis (25%) 0.45 — 0.90 0.45 — 0.90 8.3% mixture 4.15% Staph. aureus 25% (MSSA or MRSA) 0.45 0.90 — 0.450.90 — 8.3% 4.15% D-water 0.225 0.225 0.225 0.675 0.675 0.675 Totalvolume (ml) 2.70 2.70 2.70 2.70 2.70 2.70  B. Experimental samples (ml)Composition IA IIA IIIA IB IIB IIIB Final concentrations Amino acids Cys72 mM 0.225 0.225 0.225 0.225 0.225 0.225 6 mM Ieu 72 mM 0.225 0.2250.225 0.225 0.225 0.225 6 mM Ileu 72 mM 0.225 0.225 0.225 0.225 0.2250.225 6 mM Zinc Carbonate (72 mM) 0.45 0.45 0.45 0.45 0.45 0.45 12 mM Arg. Bicarbonate (44 mM) 0.45 0.45 0.45 0.45 0.45 0.45 24 mM  Staph.epidermidis (25%) 0.45 — 0.90 0.45 — 0.90 8.3% mixture 4.15% Staph.aureus 25% (MRSA) 0.45 0.90 — 0.45 0.90 — 8.3% 4.15% D-water 0.225 0.2250.225 0.225 0.225 0.225 Total volume (ml) 2.70 2.70 2.70 2.70 2.70 2.70C. Negative controls Composition 1 2 3 Final concentrations Amino acidsCys 72 mM — — — — Ieu 72 mM — — — — Ileu 72 mM — — — — Zinc Carbonate(72 mM) — — — — Arg. Bicarbonate (144 mM) — — — — Staph. epidermidis(25%) 0.45 — 0.90 8.3% mixture 4.15% Staph. aureus 25% (MSSA or MRSA)0.45 0.90 — 8.3% 4.15% D-water 1.80 1.80 1.80 Total volume (ml) 2.702.70 2.70 Arginine bicarbonate is absent in IV, V and VIDilutions of Experimental and Negative Control Samples and Inoculationsof BHI Blood Agar and CHROMagar Staph. aureus Plates

Serial dilutions from 10¹ to 10¹⁰ of each of experimental samples I, II,III, IV, V, VI and control samples 1, 2, 3 (see Table 1) were preparedwith sterile distilled water. Each dilution contained 0.1 ml of serialdiluted sample and 0.9 ml of sterile distilled water. BHI Blood agarplates were then inoculated with a mixture of 100 μl of a 10⁴ to 10¹⁰concentration of Staph. epidermidis bacteria and 100 μl of a 10⁴ to 10¹⁰sample of Staph. aureus (MSSA or MRSA) mixture (Samples I, IV andNegative Control 1) onto CHROMagar Staph. aureus plates using sterileglass bars on a turning table, respectively.

Incubation Procedures

As a first precautionary step, all agar plates were incubated for 24hours in a 37° C. incubator and examined thereafter for bacterial growthto ensure initial agar plate sterility. Plates were then inoculated withsamples taken at times 0, 24, 48 and 72 hours in succession throughoutthe 4 days of incubation. Successive inoculations consisted of thetransfer of bacterial samples from a prior incubation to a subsequentfresh sterile plate, followed by incubation at 37° C. for 24-48 hoursand subsequently repeating the process.

Colony density was scored for each of the plates as follows: between 0and 10 as 0-no colonies; 1-<10 colonies; 2-10 to 20 colonies; 3-20 to 30colonies; 4-30 to 50 colonies; 5-50 to 100 colonies; 6-100 to 250colonies; 7-250 to 500 colonies; 8->500 colonies; 9-colonies almostfused to form a layer; 10-colonies forming a bacterial layer.

Differentiation of Colonies of Staph. aureus and Staph. epidermidisDerived from Growth on BHI Blood and CHROMagar SA Plates of Samples fromIncubation Mixtures with Staph. aureus and Staph. epidermidis

Staph. aureus colonies are usually a golden yellow color and show largeand complete blood hemolytic rings around the colonies that grow on BHIBlood agar plates. Use of the coagulase serum test (test procedure ofRabbit Coagulase Plasma provided by Pro-Lab Diagnostics, Austin, Tex.USA) and Prolex Staph Xtra Latex Test (Test Protocol of Prolix™ StaphXtra Latex Kit provided by Pro Lab Diagnostics, Austin, Tex. USA) showedpositive results. On CHROMagar Staph. aureus plates, where Staph. aureuscolonies readily grow, they show, as pointed out above, a mauve color.In contrast, their counterpart, Staph. epidermidis colonies, are whiteand have no or small hemolytic rings around the colonies, when grown onBHI Blood agar plates. On CHROMagar Staph. aureus plates, Staph.epidermidis is unable to grow or able to form tiny white colonies.Coagulase serum and Prolex Staph Xtra Latex testing proved negative(i.e. no coagulation).

Inoculation of Samples Incubated in a Water Bath at 37° C. for 24 Hoursand then Inoculated onto (i) BHI Blood Agar Plates and (ii) CHROMagarStaph. Aureus Plates

Following the same serial dilution procedures, as done for the Day 1incubation period, Samples I, II, III, IV, V, VI and 1, 2, 3 werediluted serially 10⁴ to 10¹⁰ on BHI Blood agar plates. Similarly,samples of a mixture of Staph. epidermidis and Staph. aureus (I, IV andNegative Control 1) were prepared on CHROMagar Staph. aureus plates andincubated using the same procedures, as were used on Day 1, i.e.incubation at 37° C. for 24-48 hours.

Addition of Extra Ingredients to Samples, IA, IIA, IIIA and D3, IIB,IIIB Incubated as on Day 1, in a Water Bath at 37° C. for 24 Hours

-   -   Under aseptic conditions, samples, IA, IIA, IIIA and IB, IIB,        IIIB were each centrifuged and 1.35 ml of supernatant was        removed from each of samples, IA, IIA, IIIA, and 1.125 ml of        supernatant from samples, IB, IIB, IIIB, respectively.    -   The table immediately below, lists additional ingredients        introduced into samples:

TABLE 1.2 Volumes (ml) added to experimental samples Ingredients IA IIAIIIA IB IIB IIIB Cys 72 mM 0.225 0.225 0.225 0.225 0.225 0.225 Ieu 72 mM0.225 0.225 0.225 0.225 0.225 0.225 Ileu 72 mM 0.225 0.225 0.225 0.2250.225 0.225 Zinc Carbonate 0.225 0.225 0.225 — — — (72 mM) Arg.Bicarbonate 0.450 0.450 0.450 0.450 0.450 0.450 (144 mM)

Incubation of all experimental and control samples in a 37° C. waterbath was continued for another 24 hours. Total incubation time to thispoint was 48 hours.

Day 3 in the Experimental Protocol (i.e., the 48-72 Hour Time Period).

This period consisted of bacterial growth on the medium agar platesinoculated on Day 2 and incubated at 37° C., (as above), on medium agarplates for another 24 hours and preparation of samples for incubationcontinuation for another 24 hours. Bacterial growth on BHI Blood agarand CHROMagar Staph. aureus plates was then determined as before.

The next step was inoculation of samples incubated in a 37° C. waterbath for a total of 48 hours on the BHI Blood agar plates and CHROMagarStaph. aureus plates.

-   -   The same procedures of serial dilutions, as was done on Day 1,        was carried out here; i.e. all samples (I, II, III, IV, V, VI,        1, 2, 3 and IA, IIA, IIIA, IB, IIB, IIIB)    -   Inoculated 10⁴ to 10¹⁰ serial dilutions of samples on BHI Blood        agar plates and the samples of the mixture of SE and SA (I, IA,        IB, IV and Negative Control 1) on CHROMagar Staph. aureus plates        were tested by following the same procedures as was done on Day        1.    -   Plates were incubated as before at 37° C. between and for 24 and        48 hours.        Preparation of Samples for Incubation in a Water Bath at 37° C.        for 48 Hours and Followed then for a Further 24 Hours    -   Additional ingredients were added to samples of IA, IIA, IIIA        and IB, IIB, IIIB, which were each incubated in a 37° C. water        bath for a total period of 48 hours.    -   Samples IA, IIA, IIIA and samples IB, IIB, IIIB were centrifuged        as before and 1.35 ml of supernatant was removed from samples,        IA, IIA, IIIA; and 1.125 ml of supernatant was also removed from        samples, IB, IIB, and IIIB, respectively.    -   Table 1.3, below, was followed in order to serve as a guide for        adding additional ingredients into the samples:

TABLE 1.3 The (ml) volumes added to the experimental samples IngredientsIA IIA IIIA IB IIB IIIB Cys 72 mM 0.225 0.225 0.225 0.225 0.225 0.225Ieu 72 mM 0.225 0.225 0.225 0.225 0.225 0.225 Ileu 72 mM 0.225 0.2250.225 0.225 0.225 0.225 Zinc Carbonate 0.225 0.225 0.225 — — — (72 mM)Arg. Bicarbonate 0.450 0.450 0.450 0.450 0.450 0.450 (144 mM)

-   -   Incubation of all experimental and control samples in the water        bath at 37° C. was extended for another 24 hours (i.e. 72 hours        total).

Day 4 (72-96 Hours, i.e., the Last Part of the Instant ExperimentalProtocol)

Bacterial growth on medium agar plates inoculated on Day 3 was examinedand then incubated in a water bath at 37° C. for a total of 72 hours.

Examination of Bacterial Growth on BHI Blood Agar and CHROMagar Staph.Aureus Plates Inoculated on Day 3

The same methods were followed as was done on Day 4.

Inoculation of Samples Incubated at 37° C. for a Total of 72 Hours onBHI Blood Agar Plates and CHROMagar Staph. Aureus Plates

-   -   The same procedures of serial dilution were followed as was done        on Day 1 for all samples (I, II, III, IV, V, VI, 1, 2, 3 and IA,        IIA, IIIA, IB, IIB, IIIB,)    -   Inoculation of 10⁴ to 10¹⁰ serial dilutions of samples on BHI        Blood agar plates and the samples of the mixture of SE and SA        (I, IA, IB, IV and 1) on CHROMagar Staph. aureus plates were the        same as the procedures carried out on Day 1.    -   Plates were then incubated at 37° C. for 24-48 hours.

Day 5 (End of Experiment, 96 Hours Total Duration)

Examination of bacterial growth on media agar plates inoculated on Day 4and a review of the entire experiment was performed. Examination ofbacterial growth on BHI Blood agar and CHROMagar Staph. aureus platesinoculated was performed on Day 4 by following the same methods as wasdone on Day 1.

Results

Overview of the bacterial growth of all samples on the BHI Blood agarplates and on the CHROMagar Staph. aureus plates in the 72 hourexperiments reported herein are shown in Tables 1.4, 1.5 and 1.6. FIGS.1-15 depict the effect of different media on bacterial growth.Photographs showing colony growth data from which the Figures werederived are set forth as FIGS. 16-23.

TABLE 1.4 Density (1-10*) of colonies of Staphylococcus epidermidis (SE)and Staphylococcus aureus (MSSA) when incubated in media comprised of 6mM cysteine, 6 mM isoleucine, 6 mM leucine (i.e., 6 mM CIL) and 12 mMzinc carbonate, with or without 24 mM arginine bicarbonate at 37° C. for72 hours, compared with negative control (water only) Medium-Cys, Ileu,Leu, Medium-Cys, Ileu, Leu, Negative Control zinc carbonate with zinccarbonate without Medium (Water only) arginine bicarbonate argininebicarbonate Time of Times of dilution of 8.3% bacteria incubated inmedia Bacteria Incubation Plates 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵10⁶ 10⁷ SE  0 h BHI 9 9 8 8 9 9 8 8 9 9 8 8 MSSA Blood 9 9 9 9 10  9 9 910  9 9 9 Mix Agar 9 9 9 9 10  9 9 9 10  9 9 9 CHRO 9 9 9 9 10  9 9 910  9 9 9 SE/SA . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . % (~) SE 24 h BHI 9 8 7 7 8 7 6 5 5 1 1 1 MSSA Blood 9 87 5 7 3 2 1 8 7 5 4 Mix Agar 9 8 7 5 8 6 4 3 8 7 7 6 CHRO 9 8 7 5 6 4 21 8 7 7 6 SE/SA . . . . . .  5/95 10/90 . . . 70/30 60/40 80/20 . . .15/85 15/85 20/80 % (~) SE 48 h BHI 9 8 7 6 5 2 1 1 0 0 0 0 MSSA Blood 87 6 5 5 2 1 1 8 7 6 6 Mix Agar 9 8 7 6 6 2 1 1 8 7 6 5 CHRO 9 7 6 5 5 21 1 8 7 6 5 SE/SA . . . . . . 10/90 20/80 10/90 15/85 . . . . . . . . . 5/95  5/95  5/95 % (~) SE 72 h BHI 8 7 6 3 1 1 0 0 0 0 0 0 MSSA Blood 86 4 3 0 0 0 0 7 2 1 1 Mix Agar 9 7 5 3 2 0 0 0 7 5 4 2 CHRO 9 7 5 4 1 00 0 7 5 4 X SE/SA . . . 5/95  5/95 0 20/80 . . . . . . . . . . . . 0 0 .. . % (~) SE, Staph. epidermidis, MSSA, Staph. aureus (MSSA), Mix,mixture of Staph. epidermidis and Staph. aureus (MSSA), CHRO, CHROMAgarmedium plate selective for Staph. aureus, X, contamination *Scale(0-10): 0, no colony; 1, <10; 2, 10-20; 3, 20-30; 4, 30-50; 5, 50-100;6, 100-250; 7, 250-500; 8, >500; 9, colonies almost form a layer and areunable to count; 10, colonies form a layer

TABLE 1.5 Density (1-10*) of colonies of Staphylococcus epidermidis (SE)and Staphyloccus aureus (MRSA) when incubated in media comprised of 6 mMcysteine, 6 mM isoleucine, 6 mM leucine (i.e., 6 mM CIL) and 12 mM zinccarbonate, with or without 24 mM arginine bicarbonate at 37° C. for 72hours, compared with negative control (water only) Medium-Cys, Ileu,Leu, Medium-Cys, Ileu, Leu, Negative Control zinc carbonate with zinccarbonate without Medium (Water only) arginine bicarbonate argininebicarbonate Time of Times of dilution of 8.3% bacteria incubated inmedia Bacteria Incubation Plates 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵10⁶ 10⁷ SE  0 h BHI 9 9 8 7 9 8 8 8 9 8 8 8 MRSA Blood 10  9 8 8 10  9 88 9 8 8 8 Mix Agar 10  9 8 7 10  9 8 8 9 8 8 8 CHRO 10  9 8 7 10  9 8 89 8 8 8 SE/SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . % (~) SE 24 h BHI 9 9 8 8 8 8 8 8 7 6 6 6 MRSA Blood 10  99 8 7 5 5 5 9 8 8 8 Mix Agar 10  9 8 8 8 7 5 5 9 8 8 X CHRO 10  9 8 8 65 3 2 8 8 8 6 SE/SA . . . . . . . . . . . . . . . 60/40 80/20 80/20 . .. . . . . . . . . . % (~) SE 48 h BHI 9 8 7 6 6 4 3 1-3 0 0 0 0 MRSABlood 9 8 7 7 1 1 0 0 7 6 3 1 Mix Agar 9 8 7 6 1 0 0 0 7 6 5 5 CHRO 9 86 6 0 0 0 0 6 5 4 4 SE/SA . . . . . . . . . 5/95 . . . . . . . . . . . .10/90 20/80 20/80 20/80 % (~) SE 72 h BHI 8 8 7 6 5 5 4 3 1-3 0 1-3 0MRSA Blood 9 8 7 7 0 0 0 0 7 6 4 4 Mix Agar 8 8 7 6 1 1 1 1 7 7 6 6 CHRO8 8 6 5 0 1 1 0 7 6 6 6 SE/SA . . . . . . . . . . . . . . . . . . . . .. . .  5/95 10/90  5/95  5/95 % (~) SE, Staph. epidermidis, MRSA, Staph.aureus (MRSA), Mix, mixture of Staph. epidermidis and Staph. aureus(MRSA), CHRO, CHROMAgar medium plate selective for Staph. aureus, X,contamination *Scale (0-10): 0, no colony; 1, <10; 2, 10-20; 3, 20-30;4, 30-50; 5, 50-100; 6, 100-250; 7, 250-500; 8, >500; 9, colonies almostform a layer and are unable to count; 10, colonies form a layer

TABLE 1.6 Density (1-10*) of colonies of Staphylococcus epidermidis (SE)and Staphyloccus aureus (MRSA) when incubated in media comprised of 6 mMcysteine, 6 mM isoleucine, 6 mM leucine (i.e., 6 mM CIL) and 12 mM zinccarbonate, with or without 24 mM arginine bicarbonate at 37° C. for 72hours, compared with negative control (water only) Media containing 6 mMCys, 6 mM Leu, 6 mM Ileu, 12 mM zinc carbonate, 24 mM argininebicarbonate Additional same Additional 24 mM No additional above mediaadded arginine bicarbonate added medium added in 24 and 48 hours in 24and 48 hours Time of Times of dilution of 8.3% bacteria incubated inmedia Bacteria Incubation Plates 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵ 10⁶ 10⁷ 10⁴ 10⁵10⁶ 10⁷ SE  0 h BHI 9 8 8 8 9 8 8 8 9 8 8 8 MRSA Blood 10  9 8 8 10  9 88 10  9 8 8 Mix Agar 10  9 8 8 10  9 8 8 10  9 8 8 CHRO 10  9 8 8 10  98 8 10  9 8 8 SE/SA . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . % (~) SE 24 hr BHI 8 8 8 8 8 8 8 8 8 8 8 8 MRSABlood 7 5 5 5 7 5 5 5 7 5 5 5 Mix Agar 8 7 5 5 8 7 5 5 8 7 5 5 CHRO 6 53 2 6 5 3 2 6 5 3 2 SE/SA 70/30 60/40 80/20 80/20 . . . 60/40 80/2080/20 . . . 60/40 80/20 80/20 % (~) SE 48 h BHI 6 4 3 1 7 6 4 1 8 7 5 4MRSA Blood 1 1 0 0 1 0 0 0 1 1 0 0 Mix Agar 1 0 0 0 5 1 1 0 7 5 1 1 CHRO0 0 0 0 1 0 0 0 3 1 1 0 SE/SA . . . . . . . . . . . . 90/10 . . . . . .. . . 90/10 90/10 . . . . . . % (~) SE 72 h BHI 5 5 4 3 6 5 4 2 7 5 4 4MRSA Blood 0 0 0 0 0 0 0 0 0 0 0 0 Mix Agar 1 1 1 1 3 0 0 1 7 7 6 5 CHRO0 1 1 0 0 0 0 0 5 5 4 1 SE/SA . . . . . . . . . . . . 100/0  . . . . . .. . . 90/10 80/20 80/20 80/20 % (~) SE, Staph. epidermidis, MRSA, Staph.aureus (MRSA), Mix, mixture of Staph. epidermidis and Staph. aureus(MRSA), CHRO, CHROMAgar medium plate selective for Staph. aureus, X,contamination *Scale (0-10): 0, no colony; 1, <10; 2, 10-20; 3, 20-30;4, 30-50; 5, 50-100; 6, 100-250; 7, 250-500; 8, >500; 9, colonies almostform a layer and are unable to count; 10, colonies form a layerTables 1.4-1.6, above, include the following elements:

-   -   (a) Incubation of Staph. epidermidis and Staph. aureus and their        combinations in the medium containing 12 mM zinc carbonate, 24        mM arginine bicarbonate, the CIL amino acids and their controls,        showed:    -   (i) Staph. aureus (MSSA or MRSA) quickly decreased, when        incubated in the presence of arginine bicarbonate for 24 to 48        hours; all Staph. aureus organisms completely disappeared by 72        hours (see supporting FIGS. 1, 8, 16 and 19).    -   (ii) Staph. epidermidis on the other hand decreased only        slightly, when incubated with the medium containing arginine        bicarbonate during the first 24 hours of incubation and        decreased moderately or rapidly in the 48 to 72 hours thereafter        (see relevant FIGS. 1, 2, 8, 9, 16 and 19).    -   (iii) The mixture of Staph. aureus (MSSA or MRSA) and Staph.        epidermidis also showed decreases, albeit only moderately, while        being incubated in the medium containing arginine bicarbonate        for 24 hours and where approximately 60-80% of survivors were        Staph. epidermidis. The Staph. aureus/Staph. epidermidis mixture        decreased quickly after 24 hours of incubation and almost all of        the bacteria had disappeared by 72 hours (see FIGS. 3, 11, 18        and 22).    -   (iv) In the negative control, both Staph. aureus (MSSA or MRSA)        and Staph. epidermidis and the mixtures thereof incubated in        sterile D-water, showed almost no reduction in 24 to 48 hours        and very slight reduction in 48 to 72 hours (see FIGS. 1, 8, 16        and 19).    -   (b) Incubating Staph. aureus (MSSA or MRSA), Staph. epidermidis        and their combinations in a medium containing the CIL amino        acids, and zinc carbonate without arginine bicarbonate        exhibited:    -   (i) Staph. aureus (MSSA or MRSA) that showed no or slight        reduction, while incubating for 24 to 48 hours and then        decreased slightly or moderately thereafter. Staph. aureus        showed much slower reduction of its numbers in the medium        without arginine bicarbonate than when incubated in medium        containing arginine bicarbonate (see FIGS. 2, 9, 17 and 20).    -   (ii) Staph. epidermidis showed moderate to rapid reduction in        numbers during incubation for 24 hours and disappeared after 48        hours (see FIGS. 2,9, 17 and 20).    -   (iii) Within 72 hours, the mixture of Staph. aureus (MSSA or        MRSA) and Staph. epidermidis decreased moderately, while        incubating in medium without arginine bicarbonate. Also, within        72 hours, approximately 70-90% of survivors were Staph. aureus,        whereas in the mixture incubated in the medium containing        arginine, bacteria correspondingly decreased slowly in 24 hours.        About 70-75% of survivors were Staph. epidermidis and the        mixture rapidly decreased in 48 to 72 hours. Almost all bacteria        disappeared by 72 hours (see FIGS. 3, 11, 18, 22 and 23).    -   (c) The results of Staph. aureus (MRSA) and Staph. epidermidis        being incubated in the medium including 12 mM zinc carbonate, 24        mM arginine bicarbonate, the CIL amino acids, and additional        same medium or 24 mM arginine bicarbonate being added in 24 and        48 hours during 72 hours of incubation at 37° C. showed:    -   (i) Slow Staph. epidermidis reduction during the first 24 hours        and slower reduction after 48 to 72 hours, when additional same        medium was added, at 24 and 48 hours. Staph. epidermidis even        decreased, albeit more slowly, when additional 24 mM arginine        bicarbonate was added after 24 and 48 hours, whereas Staph.        aureus (MRSA) decreased, moderately to rapidly, after 48 hours        with no microbial survivors after 72 hours. There were no        differences among the incubation media and additional medium,        whether arginine bicarbonate was or was not added (see FIG. 10        and Photo 21).    -   (ii) The mixture of Staph. aureus (MRSA) and Staph. epidermidis        decreased in a similar pattern, as did Staph. epidermidis with        60% of survivors being Staph. epidermidis after 24 hours of        incubation and more than 90% Staph. epidermidis survivors after        48 to 72 hours of incubation (see FIG. 11 and Photo 23).    -   (d) Staph. aureus (MSSA or MRSA) was incubated with 12 mM zinc        carbonate, 24 mM arginine bicarbonate and the CIL amino acids        and decreased more and faster than being incubated in medium        without arginine bicarbonate. This occurred within 72 hours of        incubation, especially after 24 hours of incubation, when        compared to samples diluted 10⁴ to 10⁶ (see FIGS. 4 and 12). In        contrast, Staph. epidermidis decreased much less and more slowly        in media containing arginine bicarbonate than being incubated in        media without arginine bicarbonate, especially during 72 hours        of incubation (see FIGS. 5 and 13).    -   (e) The pH values of Staph. epidermidis, Staph. aureus (MSSA or        MRSA) and mixtures thereof, when incubated with zinc carbonate,        CIL and with or without arginine bicarbonate, and additional        same medium or 24 mM arginine bicarbonate being added at 24 and        48 hours during 72 hours of incubation at 37° C., in comparison        to a negative control (see FIGS. 7 and 15).    -   (i) pH values of SE, SA and their mixture incubated in media        containing arginine bicarbonate were stable at pH 8.3 to 8.6.    -   (ii) pH values of SE, SA and their mixture incubated in media        without arginine bicarbonate stayed at lower pH levels i.e. 6.1        to 6.8.    -   (iii) Bacteria incubated in sterile distilled water that served        as negative controls, had similar pH values, as counterpart        bacteria incubated in media without arginine bicarbonate at pH        6.0 to 6.4.

DISCUSSION

The results obtained in the experiments above demonstrated that a mediumof 12 mM zinc carbonate, 24 mM arginine bicarbonate and 6 mM CIL (i.e.,6 mM of each of cysteine, isoleucine and leucine), when incubated in awater bath at 37° C. for 72 hours, was able to bring about a decrease inboth Staph. epidermidis (SE) and Staph. aureus (MSSA or MRSA) levels(FIGS. 1 and 8). However, such a medium favored much of a reduction ofStaph. aureus (MSSA or MRSA) and did so significantly more rapidly thanreduction of Staph. epidermidis (FIGS. 2 and 9). The number of bothbacteria decreased sharply after 24 hours of incubation (FIGS. 2 and 9).This appeared to be due to substrate depletion, since addition ofarginine bicarbonate to the medium during the Staph. epidermidisincubation only decreased its numbers slightly (FIG. 10). To be noted,Staph. aureus (MRSA) showed no positive selection at all. Almost all ofthe Staph. aureus (MRSA) bacteria involved had disappeared after 48 to72 hours (FIG. 10).

In contrast (see FIGS. 4, 5, 12 and 13), when Staph. epidermidis wasincubated without arginine bicarbonate present, its numbers decreasedmuch sooner than when the medium contained arginine bicarbonate. Staph.aureus (MSSA or MRSA) showed opposite results.

This implies that the medium containing 12.0 mM zinc carbonate, 24.0 mMarginine bicarbonate and 6.0 mM CIL amino acids was able to inhibit thegrowth of Staph. aureus (MSSA or MRSA), while maintaining growth ofStaph. epidermidis. In other words and needing emphasis is that argininebicarbonate was able to support the growth of Staph. epidermidis, whilenot similarly benefiting Staph. aureus (MSSA or MRSA) at all.

As a Non-Limiting Explanation:

-   -   (1) Media containing arginine bicarbonate was able to maintain        the media pH at a constant 8.3-8.6 pH level during 72 hours of        incubation (see FIGS. 7 and 15). This was beneficial for the        growth of Staph. epidermidis, which has proven herein to be a        major bacterium for maintenance of a normal skin microflora and        for suppressing Staph. aureus (MSSA or MRSA), i.e. pathogens of        considerable concern. The medium containing zinc carbonate and        CIL, but with no arginine bicarbonate present, had a pH between        6.1 and 6.8 (see FIGS. 7 and 15), which evidently was able to        inhibit the growth of Staph. aureus (MSSA or MRSA) slightly to        moderately (see FIGS. 6 and 14). But, it was not able to        strongly inhibit Staph. aureus (MSSA or MRSA), in a medium        containing arginine bicarbonate (see FIGS. 6 and 14 vs. 4 and        12). In contrast, Staph. epidermidis was quickly reduced in this        medium (FIGS. 5 and 13). This would most importantly imply that        a reason for this is that the alkaline pH (8.3-8.6), which        promoted the growth of Staph. epidermidis, and its anti-Staph.        aureus effectiveness, resulting in reduction of the growth of        Staph. aureus (MSSA or MRSA).    -   (2) Evidently, as explanation, the pH may not have been the only        factor to affect the survival of Staph. epidermidis and Staph.        aureus.

Although the overall pH of the medium (zinc carbonate, argininebicarbonate and CIL) and additional same medium or 24 mM argininebicarbonate being added at 24 and 48 hours during 72 hours ofincubation, was maintained at pH 8.3-8.6; it showed remarkably well thatas more arginine bicarbonate was added to the medium, the density ofStaph. epidermidis that was ultimately obtained was increased.Nonetheless and most importantly, this indicated that argininebicarbonate can play a significant enhancement role in the growth ofStaph. epidermidis and that this effect may be largely but not solelydue to the elevated and sustained pH favored by the presence of argininebicarbonate.

In contrast, Staph. aureus (MSSA or MRSA) incubated in the mediumcontaining zinc carbonate, CIL and no arginine bicarbonate or in asterile distilled water negative control (both of which show a pH in therange of 6.0-6.8) showed almost no reduction in growth after 72 hours ofincubation in distilled water (see FIGS. 1, 7, 8 and 15). However, therewas moderate reduction during incubation for 72 hours in a mediumcontaining zinc carbonate, and CIL without arginine bicarbonate (seeFIGS. 6 and 14). Accordingly, one can conclude that zinc carbonate is animportant ingredient for suppression of Staph. aureus (MSSA and MRSA)growth, and plays thereof a significant inhibitory role as well.

The present invention is not limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description. Patents, patent applications, publications,product descriptions, and protocols are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

REFERENCES

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1. An antibacterial composition, comprising: a. arginine, or a saltthereof; b. a zinc salt; c. optionally, a buffer sufficient to maintainthe pH of said composition at 6.0 or greater upon topical application;and d. a physiologically-acceptable carrier suitable for topicalapplication, said composition being capable of inhibiting the growthand/or metabolism of Staphylococcus aureus to a greater extent than thegrowth and/or metabolism of Staphylococcus epidermidis.
 2. Thecomposition of claim 1, further comprising phenylalanine.
 3. Thecomposition of claim 1, said composition being capable of inhibiting thegrowth and/or metabolism of malodor-generating microbiota present in thecutaneous regions of a subpart of the human body.
 4. The composition ofclaim 3, wherein said cutaneous regions comprise the axilla, foot-websand nasal atrium.
 5. The composition of claim 1, wherein said zinc saltis selected from zinc carbonate and zinc bicarbonate.
 6. The compositionof claim 1, wherein said arginine, or a salt thereof is selected fromarginine, arginine carbonate and arginine bicarbonate.
 7. Thecomposition of claim 1, wherein said zinc salt is zinc carbonate andsaid arginine salt is arginine bicarbonate.
 8. The composition of claim1, wherein said composition is provided as a topical formulationselected from the group selected from soap, spray, drop, aerosol,powder, roll-on, lotion, cream, stick, solution, sachet, colloidalsuspension, film, patch and ointment.
 9. (canceled)